Digestion with NEBNext dsDNA Fragmentase (M0348)
Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community.
Tip: Adequate mixing of NEBNext dsDNA Fragmentase is important for the success of this reaction. NEBNext dsDNA Fragmentase should be vortexed for 3 seconds immediately prior to use.
For tough digestions, add 1 μl of 200 mM MgCl2 to the reaction. Additional MgCl2 can be added if necessary.
The protocol listed below is for fragmentation of 5 ng–3 μg of DNA.
- Vortex NEBNext dsDNA Fragmentase for 3 seconds, quick spin and place on ice.
- Combine the following components in a sterile PCR tube and vortex:
DNA (5 ng–3 μg) 1–16 μl 10X Fragmentase Reaction Buffer v2 2 μl Sterile Water variable Final Volume 18 μl
- Add 2.0 μl dsDNA Fragmentase and vortex the mixture for 3 seconds.
- Note: Fragmentase is very viscous and should be pipetted slowly. If the enzyme has been sitting for several minutes vortex it again before adding to the sample.
- Incubate at 37°C for the recommended times below to generate the desired fragment size. To determine the exact incubation time for a given sample type, a time course study should be performed.
Desired Fragment Size (bp) Incubation Time (min) 50–200 25–35 200–1,000 15–25 1,000–2,000 10–15
- Add 5 μl of 0.5 M EDTA to stop the reaction.
- Clean up the fragmented DNA with column purification or using SPRI beads. If using SPRI beads, it is recommended to dilute the sample 1:1 with sterile water for easier handling of the sample and faster collection of the beads to the magnet.
End Repair: Clean up the fragmented DNA then proceed with desired DNA end repair protocol.
Polyacrylamide Gel Analysis: Clean up the fragmented DNA prior to loading the samples on a PAGE gel.
Long Term Storage: Clean up the fragmented DNA prior to long term storage.
Agarose Gel Size Selection/Analysis: Samples can be loaded directly on to an agarose gel. It is not necessary to clean up the reactions prior to loading.